Pharmaceutical composition containing glur2-lacking ampar antagonist for preventing or treating psychiatric illnesses

ABSTRACT

A pharmaceutical composition, comprising a GluR2-lacking AMPAR antagonist as an active ingredient, is effective for preventing or treating mental diseases selected from the group consisting of posttraumatic stress disorder (PTSD), drug addiction, and phobia.

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition forpreventing or treating mental diseases, comprising a GluR2-lacking AMPARantagonist as an active ingredient and a use thereof.

BACKGROUND OF THE INVENTION

The treatment of posttraumatic stress disorder (PTSD) has been mostlyfocused on the alleviation of the symptoms by counseling such asexposure therapy and cognitive restructuring. Although medicationtherapy is available, only indirect palliatives such as antidepressantsare used, which makes it hard to treat critical PTSD patients.

Meanwhile, although there are various methods for treating patientssuffering from drug addiction or phobia, repeated memory renewal afterthe treatment is the most prominent clinical difficulty. Therefore,there exists a need to investigate physiological mechanism of memoryrenewal for preventing renewal of drug addiction/fear memory.

The α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor(AMPAR) is an ionotropic glutamate receptor that mediates fast synaptictransmission in the central nervous system (CNS). AMPARs are composed offour types of subunits, designated GluR1, GluR2, GluR3, and GluR4. Ca2+permeability of AMPAR depends on the constitution of the subunits, i.e.,receptors having GluR2 subunit show low Ca2+ permeability whilereceptors having no GluR2 subunit show high Ca2+ permeability (TrendsNeurosci. 16, 359-365, 1993; Annu. Rev. Neurosci. 17, 31-108, 1994;Prog. Neurobiol. 54, 581-618, 1998).

GluR2-lacking AMPAR antagonists, mostly derivatives of spermine, havebeen reported to be useful for treating epilepsy and cerebral infarction(Yoneda et. al., Bioorg Med Chem. 11(10), 1261-1264, 2001). However, ithas not been reported whether GluR2-lacking AMPAR antagonists may beeffective in the treatment of PTSD, drug addiction or phobia.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a pharmaceuticalcomposition for preventing or treating mental diseases, comprising aGluR2-lacking AMPAR antagonist as an active ingredient.

There is provided a pharmaceutical composition for preventing ortreating mental diseases selected from the group consisting ofposttraumatic stress disorder (PTSD), drug addiction, and phobia,comprising a GluR2-lacking AMPAR antagonist as an active ingredient.

Further, it is another object of the present invention to provide a useof GluR2-lacking AMPAR antagonists for preventing or treating mentaldiseases selected from the group consisting of PTSD, drug addiction, andphobia in a subject.

A pharmaceutical composition comprising a GluR2-lacking AMPAR antagonistas an active ingredient is useful for preventing or treating mentaldiseases in a subject since the antagonist prevents the necrosis ofbrain cells by inhibiting accumulation of calcium (Ca²⁺), zinc (Zn²⁺),etc. in the neurons and reduces neural activity of the correspondingbrain regions related to the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings, which respectivelyshow that:

FIG. 1: GluR2-lacking AMPAR accumulation in the amygdala synapse byrepeated retrieval of fear memory;

FIG. 2: suppression of amygdala synaptic responses by a GluR2-lackingAMPAR antagonist, NASPM, in the experimental groups of memory extinctionand memory renewal; and

FIG. 3: concentration-dependent blocking of the renewal of fear memoryby the administration of a GluR2-lacking AMPAR antagonist, NASPM, intothe amygdala.

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutical composition of the present invention for preventingor treating mental diseases comprises a GluR2-lacking AMPAR antagonistas an active ingredient.

The mental diseases include posttraumatic stress disorder (PTSD), drugaddiction, and phobia.

The composition of the present invention comprising a GluR2-lackingAMPAR antagonist as an active ingredient prevents the necrosis of braincells by inhibiting the accumulation of calcium (Ca²⁺), zinc (Zn²⁺),etc. in the neurons and reduces neural activity of the correspondingbrain regions related to the disease, thereby suppressing the underlyingbehaviors of the mental diseases such as PTSD, drug addiction, andphobia.

The GluR2-lacking AMPAR antagonist may be N-naphthylspermine (NASPM),philanthotoxin, Joro spider toxin orN-(4-hydroxyphenylpropanoyl)-spermine (NHPP-spermine).

Further, the composition of the present invention comprising theGluR2-lacking AMPAR antagonist may further comprise other ingredientssuch as vitamins, minerals, and blood circulation improving agents.

Examples of suitable vitamins include vitamin B1, B2, B6, B12, C,calcium pantothenate, nicotinamide, folic acid, and biotin.

Examples of suitable minerals include zinc, iron, calcium, andmagnesium.

It is preferred to employ the vitamins and minerals in combination forenhancing their biological activities. Preferably, a vitamin or amineral may be included in the composition in an amount of 0.1 to 10 wt% based on the total weight of the composition. More preferably, vitaminB1, B2, and C may be included in amounts of 0.01 to 3 wt %, 0.01 to 3 wt%, and 0.01 to 4 wt %, respectively, based on the total weight of thecomposition.

Examples of suitable blood circulation improving agents aregamma-linolenic acid, EPA, and tocopherol.

The blood circulation improving agents may be employed in combination inorder to enhance the blood circulation effect and biological activities.Preferably, the blood circulation improving agent is tocopherol, and itmay be used in an amount of 5 to 30 wt % based on the total weight ofthe composition.

Further, the composition of the present invention may further comprise asolvent which is capable of dissolving all the ingredients including theGluR2-lacking AMPAR antagonist, vitamins, minerals, and bloodcirculation improving agents. Examples of suitable solvents are waterand ethyl alcohol.

Meanwhile, the composition of the present invention is preferablyprocessed into pharmaceutical preparations, together with apharmaceutically acceptable carrier, in accordance with the conventionalmedicament preparation methods. Examples of suitable carriers arelactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate,calcium silicate, cellulose, methyl cellulose, microcrystallinecellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and a mineral oil.

Further, the composition of the present invention may further comprisefillers, anti-agglomerating agents, lubricating agents, wetting agents,flavoring agents, emulsifiers or preservatives.

The composition of the present invention may be formulated so as toprovide a quick, sustained or delayed release of the active ingredientafter administration to a subject by any of the known methods. Theformulation may be in the form of a tablet, pill, granule, powder,sachet, elixir, suspension, emulsion, solution, syrup, aerosol, soft andhard gelatin capsule, sterile injectable solution, sterile packagedpowder, and others. In the preparation of the formulation such as agranule and liquid, it may be desirable to add thereto a naturalflavoring agent or natural juice having the flavor of plum, lemon,pineapple or herb, a natural coloring agent such as chlorophyllin, asweetening agent such as fructose, honey, sugar alcohol, sucrose or anacidifier such as citric acid and sodium citrate.

Further, the composition of the present invention can be administratedby various routes such as oral administration or injection.

An effective daily dose of the composition of the present invention mayrange from about 0.01 to 20 g/kg. However, it should be understood thatthe amount of the active ingredient actually administered ought to bedetermined in consideration of various relevant factors including theage, sex, the weight of the patient to be treated, and the severity ofthe symptom of the disease. The daily dose should not be intended tolimit the scope of the invention in any way.

Further, the present invention also provides a use of GluR2-lackingAMPAR antagonist for preventing or treating mental diseases selectedfrom the group consisting of PTSD, drug addiction, and phobia in asubject.

The subject may be a mammal, e.g., human.

The GluR2-lacking AMPAR antagonist may be N-naphthylspermine (NASPM),philanthotoxin, Joro spider toxin orN-(4-hydroxyphenylpropanoyl)-spermine (NHPP-spermine).

The following Examples are intended to further illustrate the presentinvention without limiting its scope.

EXAMPLE 1 Accumulation of GluR2-Lacking AMPAR in the Amygdala Synapse byRepeated Retrieval of Fear Memory

Tests were conducted to examine whether GluR2-lacking AMPAR accumulatesin the amygdala synapse by repeated retrieval of fear memory.

Example 1-1 Fear Memory Learning, Memory Extinction, and Memory Renewal

White rats, 4-5 weeks old (Sprague-Dawley Rat, Male; Orient Bio Inc.,Korea) were used in the following experiments. All rats were kept whileallowing free access to food and water under a 12 hour cycle of lightand dark (the light was turned off at 9am).

For fear memory learning, the rats were exposed to sound stimuli of asingle tone (2.8 kHz, 85 dB) for 30 seconds, and subjected to anelectric foot shock (1.0 mA) for 1 second at the end of said 30-secondperiod. The sound stimuli and the electric foot shock were repeated 3times at 100 second intervals. 1 minute later after the last electricfoot shock, the rats were returned to their home cages. On day 2, fearmemory learning was performed in the same way.

From day 3, memory extinction was performed in a context different fromthat of the fear memory learning On day 4, the rats were exposed to onlysound stimuli 20 times at 100 second intervals without an electric footshock. On days 5 and 6, the sound stimuli were repeated 15 times at 100second intervals.

On day 7, memory renewal test was performed. The rats experienced memoryextinction were placed in the place of the fear memory learning for 10minutes, and then, memory renewal was induced by exposing the rats tosound stimuli for 30 seconds.

Example 1-2 Preparation of Brain Slices

The brains of the rats of the group of memory extinction and memoryrenewal were extracted and placed in a modified artificial cerebrospinalfluid (aCSF). The composition of the modified artificial cerebrospinalfluid used was as follows: 175 mM sucrose, 20 mM NaCl, 3.5 mM KCl, 1.25mM NaH₂PO₄, 26 mM NaHCO₃, 1.3 mM MgCl₂, and 11 mM D-(+)-glucose(Sigma-Aldrich Co., USA). About 3 minutes later, the brains were slicedinto 0.3 mm thick slices using a vibroslicer (HA752, CampdemInstruments, Loughborough, UK). The brain slices containing amygdalawere cultured in a artificial cerebrospinal fluid (120 mM NaCl, 3.5 mMKCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 1.3 mM MgCl₂, 2 mM CaCl₂, 11 mMD-(+)-glucose) containing 95% O₂/5% CO₂ at room temperature for over 1hour. The cortex overlying the amygdala was removed just before takingit to the equipment for measuring the synaptic reaction.

Example 1-3 Measurement of Synaptic Efficacy Using Whole-CellPatch-Clamp

Synaptic responses were measured in 32-34° C. artificial cerebrospinalfluid using Axopatch 200A or Multiclamp 700A (Molecular Devices, CA). Apatch-pipette solution based on potassium (K) was prepared whichconsisted of the following: 120 mM K-gluconate, 0.2 mM EGTA, 10 mMHEPES, 5 mM NaCl, 1 mM MgCl₂, 2 mM Mg-ATP, and 0.3 mM Na-GTP(Sigma-Aldrich Co., USA).

To confirm whether the neuron recorded is a principal neuron, the shapeof the cell and spike-frequency adaptation of the action potentialinduced by a current injection was examined. Picrotoxin (0.1 mM,Sigma-Aldrich Co., USA) was added to the artificial cerebrospinal fluidto block inhibitory inputs to principal neurons. The pipette resistancesranged from 2.5 to 3.5 Mohm. The pipette series resistance was monitoredthroughout the experiments, and if it changed by >20% the data werediscarded. All the experiments were performed within 3.5 hours afterpreparing the brain slices.

Example 1-4 Measurement of Rectification Index (RI)

The rectification index (RI) was calculated as the ratio of synapticresponses at membrane potentials of −60 mV and +40 mV, which increasedas the GluR2-lacking AMPAR accumulates.

The internal solution contained spermine (0.1 mM, Sigma Aldrich Co.,USA). The reversal potential (Erev) was measured in each experiment. RI(Erev +40 mV/Erev −60 mV) was compared at 20 minutes after the start ofwhole-cell recordings to ensure complete diffusion of exogenous spermineinto the cell interior, and D-AP5 (Tocris, USA) was applied 5 minutesbefore the RI estimation to isolate AMPAR-mediated excitatorypostsynaptic currents (EPSCs) at positive potentials. The RI at themembrane potentials of −60 mV and 40 mV were measured 6 times,respectively. The average of RI values was used to determine the RI ofthe neuron.

Example 1-5 Results

The rats of the control group which did not experience fear memorylearning showed RI of 1.5, which shows that the synaptic response sizeis in direct proportion to the membrane potential size (Tsvetkov et al.,Neuron, Vol. 34, 289-300, Apr. 11, 2002). In comparison, the rats of theexperimental groups of memory extinction and memory renewal whichexperienced repeated retrieval of fear memory showed higher RI values.Further, the rats of memory renewal group showed significantly higher RIvalues than those of memory extinction group (see FIG. 1). This suggeststhat GluR2-lacking AMPAR accumulates in the amygdala synapse as fearmemory repeats and recurs.

EXAMPLE 2 Suppression of the Synaptic Reaction By GluR2-Lacking AMPARAntagonist, NASPM

0.3 mm thick brain slices were prepared from the rats which experiencedfear memory learning, memory extinction, and memory renewal inaccordance with the method described in Examples 1-1 and 1-2.

The thalamic afferent fibers connecting thalamus to the lateral amygdalawere stimulated using concentric bipolar electrode (MCE-100, RhodesMedical Instruments, CA, USA) in order to induce synaptic responses. Inorder to examine the basal response value of the amygdala synapse, thesize of synaptic response was measured for 10 minutes, 200 μM of NASPM(N-naphthylspermine, Sigma-Aldrich Co., USA) dissolved in a artificialcerebrospinal fluid was administered, and the size of synaptic responseswas measured.

As a result, the synaptic response of the amygdala was inhibited by 200μM of NASPM in the experimental groups of memory extinction and memoryrenewal (see FIG. 2). The results of the control group, obtained byVadim Bolshakov of Harvard university (Tsvetkov et al., 2002), show thatthe inhibition by NASPM is subtle in case of the rats which did notexperience fear memory learning The result obtained by NASPM proves thatGluR2-lacking AMPAR accumulates in the amygdala synapse as fear memoryrelapses.

EXAMPLE 3 Blocking Effect Of Fear Memory Renewal By GluR2-Lacking AMPARAntagonist, NASPM

An in vivo experiment was performed in order to examine whether NASPMadministered in the amygdala blocks fear memory renewal.

Example 3-1 Fear Memory Learning, Memory Extinction and Memory Renewal

The experiment was performed using the rats which experienced fearmemory learning, memory extinction and memory renewal in accordance withthe method described in Example 1-1. White rats, 8-9 weeks old(Sprague-Dawley Rat, Male; Samtako, Korea), were used.

Example 3-2 Administration of NASPM

The rats were anesthetized by intraperitoneal injection of pentobarbitalsodium (50 mg/kg; Entobal, Hanlim Pharma, Co., Ltd., Korea). After fullyanesthetized, the rats were mounted on a stereotaxic apparatus (DavidKopf Instruments, Tujunga, Calif., USA), and then, stainless steelcannulas of 26 gauge (Model C315G, Plastic Products, Roanoke, Va., USA)were implanted bilaterally into the amygdala (AP; −0.3 mm, ML; +/−5.15,DV; −7.0 mm) by operation. A 32 gauge dummy cannula was inserted intoeach cannula to prevent clogging. After a recovery period of at least 1week, the rats were subjected to fear memory learning, memory extinctionand memory renewal tests. Then, the brain slices of 0.08 mm thicknesswere prepared and stained with cresyl violet (Sigma-Aldrich Co., USA)cannula in order to confirm whether the cannulas were exactly implantedin the amygdalas. 40 μg and 10 μg of NASPM (Sigma-Aldrich Co., USA) wererespectively dissolved in 0.0005 ml of normal saline, and then, thesolutions were respectively administered to the rats using a 0.01 mlhamilton syringe at a rate of 0.00025 ml/min through said cannulas, 15minutes before measuring fear memory.

Example 3-3 Measurement of the Fear Reaction

The duration that the rats showed freezing response while exposed to thesound stimuli was measured in order to examine remaining memory (Kim et.al., PNAS, 104(52), 20955-20960, 2007). Conditioned freezing was definedas immobility except for respiratory movements, and was quantified bytrained observers that were blind to the experimental groups.

Example 3-4 Results

In comparison with the control group, the experimental groupadministered with 40 μg of NASPM, a GluR2-lacking AMPAR antagonist,showed a remarkably less fear responses, while the group administeredwith 10 μg of NASPM showed an insignificant effect (see FIG. 3). Theseresults show that GluR2-lacking AMPAR accumulated in amygdala synapsesby the repeated fear memory recall plays an important role in fearmemory renewal. In addition, NASPM, a GluR2-lacking AMPAR antagonist,blocks the fear memory renewal in a concentration-dependent manner, whenadministered in the amygdala.

While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made to the invention by those skilled in the artwhich also fall within the scope of the invention as defined by theappended claims.

1. A pharmaceutical composition for preventing or treating mentaldiseases selected from the group consisting of posttraumatic stressdisorder (PTSD), drug addiction, and phobia, comprising a GluR2-lackingAMPAR antagonist as an active ingredient.
 2. The pharmaceuticalcomposition of claim 1, wherein the GluR2-lacking AMPAR antagonist isselected from the group consisting of N-naphthylspermine (NASPM),philanthotoxin, Joro spider toxin, andN-(4-hydroxyphenylpropanoyl-spermine (NHPP-spermine).
 3. A use ofGluR2-lacking AMPAR antagonist for preventing or treating mentaldiseases selected from the group consisting of posttraumatic stressdisorder, drug addiction, and phobia.
 4. The use of claim 3, wherein theGluR2-lacking AMPAR antagonist is selected from the group consisting ofN-naphthylspermine, philanthotoxin, Joro spider toxin, andN-(4-hydroxyphenylpropanoyl)-spermine.